Current two-photon (2P) confocal imaging systems are built around traditional confocal microscope scan heads that use galvanometers to control laser beam scanning. This technology significantly limits scanning speed. Building on recent developments of acousto-optical deflector (AOD)-based 2P microscopes we will test the feasibility of developing a commercially available AOD-based 2P imaging system. Success will permit scientists to use imaging speeds ranging from those available on current commercial systems up to 30 frames per second. Our proposal includes 4 specific aims. Specific Aim I: We will build an alpha version 2P-AOD microscope that will operate with an ultra-fast pulse laser excitation source. We will modify a recently developed 2P microscope, the Ultima, which we have developed in the past year, for AOD scanning and will construct scan control and signal conditioning circuitry to provide control and acquisition software for high-speed 2P imaging. Specific Aim II: We will extend the recent development of 'Prairie View' software, galvanometer-based confocal and two-photon (2P) software, to control the 2P-AOD microscope. Specific Aim III: We will compare the optical resolution of the 2P-AOD microscope with that of current galvanometer-scanning 2P systems (a Bio-Rad-based system, a Nikon-based C1 system and the Prairie Ultima). Specific Aim IV: We will test the performance of the 2P-AOD microscope in biological applications. Success in this project will provide the scientific community with an innovative microscope that will enhance its ability to perform live cell imaging in a range of scientific fields, including cell biology, neuroscience and regenerative medicine, and allow innovative studies in such areas as synaptic transmission, learning and memory, and regenerative medicine and stem cell biology.